Mating behavior induces a complex set of physiological response in most female mammals that are essential for successful reproduction. The central nervous system (CNS) directs mating behavior, and then transforms the neural input as a result of mating into endocrine function. The basic hypothesis being investigated in this proposal is that mating exposes a neuronal clock that results in twice-daily prolactin (PRL) surges necessary for maintenance of pregnancy. The overall goal is to characterize the CNS events that participate in these PRL surges by identifying the neuronal pathways and neuronal phenotypes responsible for their occurrence. This will be investigated in a rat model in using natural mating rather than artificial vaginal-cervical stimulation because the consequences differ, including the development of a conceptus and secretions of placental hormones. To measure neuronal activation, the appearance of the nuclear fos protein will be examined. This approach is possible because expression of the immediate early gene, c-Fos, occurs quickly upon neuronal firing. Our preliminary experiments revealed neuronal activity was strongly correlated with PRL surges in specific regions of the preoptic area and dorsomedial nucleus. Given the major roles serotonergic, opioidergic and dopaminergic pathways play in PRL regulation, these are likely candidates thaat relay expression of the clock to the median eminence and pituitary to drive PRL surges. Another major player may be vasoactive intestinal peptide (VIP), recognized as a neuromodulator of PRL, secretion and dominant peptide in the suprachiasmatic nucleus, location of the clock driving circadian rhythms. Specific Aim 1 will identify the locations of neurons which participate in the regulation of PRL surges, including the direct response to mating and after the daily surges are established. Experimental manipulation of the PRL surges will be done to demonstrate the degree of dependence to the surges on specific patterns of neuronal activity. Specific Aim 2 will identify and characterize the specific neuronal pathways and their projections that program mating-induced PRL surges. Specific antogonists will be used to block neurotransmitters or as a means of establishing their participation in the pathway. Tract-tracing immunocytochemistry, in situ hybridization, and sterotaxic placement of cells and hormones are major techniques that will be used in this project. An understanding of the essential clock-controlled genes that drives the overt circadian PRL rhythm during pregnancy will provide insights into the components of important neural and endocrine rhythms in humans that are critical to homeostasis and reproduction.